| RRC ID |
34500
|
| 著者 |
Sánchez-Hidalgo M, Pascual J, de la Cruz M, Martín J, Kath GS, Sigmund JM, Masurekar P, Vicente F, Genilloud O, Bills GF.
|
| タイトル |
Prescreening bacterial colonies for bioactive molecules with Janus plates, a SBS standard double-faced microbial culturing system.
|
| ジャーナル |
Antonie Van Leeuwenhoek
|
| Abstract |
Despite the availability of many culture-based antibiotic screening methods, the lack of sensitive automated methods to identify functional molecules directly from microbial cells still limits the search for new biologically active compounds. The effectiveness of antibiotic detection is influenced by the solubility of the assayed compounds, indicator strain sensitivity, culture media and assay configuration. We describe a qualitative high throughput screening system for detecting cell-perturbing molecules from bacterial colonies employing two opposed agar layers sequentially formed in prototype Society for Biomolecular Screening (SBS) plates, named Janus plates. Direct assay of microbial colonies against target organisms in opposed agar layers overcomes some of the limitations of agar overlay methods. The system enables the rapid detection of extracellular cell-perturbing molecules, e.g., antibiotics, excreted directly from environmental isolates. The source bacterial colonies remain separate from the target organism. The growth layer is prepared and grown independently, so environmental strains can be grown for longer intervals, at temperatures and in media that favor their growth and metabolite expression, while the assay layer with pathogens, usually requiring nutrient-rich medium and elevated temperatures, are added later. Colonies to be tested can be precisely arrayed on the first agar surface, thus avoiding dispersion and disturbance of potential antibiotic-producing colonies by overlaying agar with the target strain. The rectangular SBS configuration facilitates factorial replication of dense microbial colony arrays for testing with multiple assays and assay conditions employing robotic colony pickers and pin tools. Opposed agar layers only slightly reduced the effectiveness for detecting growth inhibition from pure antibiotics compared to single-layer agar diffusion assays. The Janus plate enabled an automation-assisted workflow where a lone operator can effectively identify and accumulate bioactive soil bacterial strains within a few weeks. We also envisage the method's utility for functional prescreening colonies of clones from genomic and metagenomic libraries or improved strains originating from mutagenized cells.
|
| 巻・号 |
102(2)
|
| ページ |
361-74
|
| 公開日 |
2012-8-1
|
| DOI |
10.1007/s10482-012-9746-7
|
| PMID |
22562433
|
| PMC |
PMC3397223
|
| MeSH |
Anti-Bacterial Agents / metabolism*
Bacteria / growth & development
Bacteria / metabolism*
Colony Count, Microbial / instrumentation
Colony Count, Microbial / methods*
Drug Evaluation, Preclinical / instrumentation
Drug Evaluation, Preclinical / methods*
High-Throughput Screening Assays / instrumentation
High-Throughput Screening Assays / methods*
|
| IF |
2.674
|
| 引用数 |
7
|
|
WOS 分野
|
MICROBIOLOGY
|
| リソース情報 |
| 一般微生物 |
JCM 4073
JCM 4297
JCM 4523
JCM 4526
JCM 4667
JCM 4775 |
